The fatigue life of preloaded bolts depends on the additional dynamic loading of the bolts, which depends on the size and location of the working force application. Due to the eccentricity of the working force, there is an additional bending load on the bolts, which has a significant effect on fatigue. In this study, the influence of the additional axial and bending load of bolts on fatigue is investigated analytically, numerically and experimentally for four different flange joints. Comparison of the results shows that the analytical method overestimates the additional bolt stresses at low working load, mainly due to the extremely large contribution of bending stress. As the working load increases, the differences between the two methods decrease, but only for the reason that the analytical method can only linearly scale the overestimated results at lower working load, whereas the FEM analysis shows a progressive increase of the additional stress in the bolt at higher working loads due to the spreading of the flange. Experimental verification confirmed that the FEM analysis can be considered as a reference method. By using a high washer, the critical point of the bolt is shifted from the first contact of the bolt thread with the nut thread to the transition of the shank to the bolt head. The reduced notch effect and larger cross-section at this point greatly increases the fatigue life of the bolt.